This invention relates to a method of enriching for and isolating subpopulations of epithelia cells, isolation of keratinocyte stem cells, to keratinocyte stem cells and uses for keratinocyte stem cells.
In common with other rapidly renewing tissues such as the haemopoietic system and the intestinal epithelia, the human epidermis is in a process of constant regeneration. Terminally differentiated cells lost continuously from the skin surface, are replaced by an intricate and highly regulated proliferative process within the basal layer of the epidermis. Stem cells in these rapidly renewing tissues are the earliest progenitors of a hierarchy of proliferative cells which are ultimately responsible for the generation of all mature cells for the lifetime of an individual (Lajtha, 1979). In murine epidermis, this process is achieved by two kinetically distinct subpopulations: (a) keratinocyte stem cells (KSC) which represent a minor subpopulation of relatively quiescent cells, defined by their great proliferative potential and an unlimited capacity for self renewal, identified as slow-cycling, 3H-Tdr label-retaining cells; and (b) transit amplifying (TA) cellsxe2x80x94the progeny of the stem cells, with a limited proliferative capacity identified as a pool of rapidly proliferating cells that are lest from the basal layer to terminal differentiation within 4-5 days (Potten, 1983: Morris et al, 1985; MacKenzie and Bickenbach, 1985; Potten, 1986; Bickenbach et al, 1986). In addition, a third Subpopulation of basal keratinocytes representing post-mitotic differentiating cells in the early stages of keralinisation can also be identified (Potten, 1983; Morris et al, 1985; MacKenzie and Bickenbach, 1985; Potten, 1986; Bickenbach et al, 1986; Christophers, 1971; Allen and Potten, 1974). Human epidermis has similar populations.
Given that all proliferative activity in the human epidermis is restricted to the basal layer, this is presumably where the stem cells and TA cells reside. It has also been established that the hair follicle can act as an important reservoir of epidermal stem cells, and that cells within the bulge region have extensive proliferative potential. Physiological cell renewal in interfollicular epidermis however, is most likely to be achieved by stem cells and TA cells within the basal layer. However there are no molecular markers that distinguish between basal keratinocytes that have made a commitment to differentiate (TA cells) and immature stem cells.
In the haemopoietic system, multilineage reconstituting stem cells can be physically separated from committed progenitor cells (analogous to the TA cells of the epidermis). based upon differences in their expression of cell surface markers (Civin et at, 1984; Spangrude et al, 1988; Berenson et al, 1991; Terstappen et al, 1991; Baum et al, 1992). Clearly the availability of appropriate cell surface markers on basal epidermal cells would greatly facilitate the isolation and characterisation of human KSCs. However, the cell surface antigenic phenotype of these cells remains relatively poorly defined.
One of the best studied classes of cell surface molecules expressed by keratinocytes are the integrin superfamily of cell adhesion receptors. Integrins are heterodimeric cell surface glycoproteins that primarily mediate the attachment of basal keratinocytes to extracellular matrix proteins found in the basement membrane, but can also mediate intercellular adhesion. In vivo, basal keratinocytes express the xcex21 integrins xcex13xcex21 and as well as the integrin xcex16xcex24 (Peltonen et at, 1989; Carter et al, 1990a Carter et al, 1990b). Important evidence for proliferative heterogeneity in human basal keratinocytes has been provided by recent work using a fluorescence activated cell sorting (FACS) approach, demonstrating that both cultured and primary human foreskin keratinocytes could be separated into cells with high levels of xcex21 integrin (xcex21 bright) which had a high plating efficiency assayed after two weeks in culture, compared to those keratinocytes with low levels of this integrin (Jones and Watt, 1993; Jones et al, 1995). Furthermore, bright keratinocytes were shown to be capable of generating an epithelial sheet when grafted onto mice, suggesting that this fraction of the basal layer contain KSCs (Jones et al, 1995).
In vivo studies suggest that epidermal stem cells constitute between 1%-10% of the basal layer depending on the methodology used (Morris et al, 1985; MacKenzie and Bickenbach, 1985; Bickenbach et al, 1986; Potten and Hendry, 1973; Morris and Potten, 1994). Since approximately 40% of the basal layer in human foreskin exhibits high levels of xcex21 integrin in vim (Jones et at, 1995) it is highly likely that basal keratinocytes with this phenotype contain both the KSC population and a significant number of TA cells and therefore there are drawbacks in the use of cells enriched for high level expression of xcex21.
An object of one aspect of the invention is to generate a more purified population of keratinocyte stem cells than has been achieved by prior art methods. An object of a further aspect of the present invention is to provide methods for purifying subpopulations of epithelial cells.
A strategy for distinguishing between the TA cells and the KSCs of the epidermis based on the use of two cell surface antigens has been shown to be effective. In view of functional data demonstrating the role of integrin xcex16xcex24 in mediating adhesion of basal keratinocytes to the basement membrane via hemi-desmosomes (Sonnenberg et al, 1991; Dowling et at, 1996; Georges-Labouessee et al, 1996; Van-der-Neut et al, 1996) it was hoped that this integrin may provide a suitable marker for epidermal stem cells since these cells are permanently anchored to the basement membrane.
It is now shown that while basal keratinocytes expressing low levels of xcex16xcex24 represent a subpopulation of post-mitotic, differentiating keratinocytes, this integrin is expressed at high levels on both the KSC and TA cells. Thus this cell surface marker alone, cannot be used to separate KSCs from TA cells to a high degree of purity but can do so to a degree of purity higher than where xcex21 integrin is used.
It is the finding of the inventors that enrichment for human KSCs to a high degree of purity can be successfully achieved on the basis of a second cell surface component whose expression is proliferation-related in conjunction with xcex16xcex24 integrin. The experiments conducted to date have used transferrin receptor as the cell surface component that is proliferation related. It is also suggested that sufficient purification should be achievable where another marker capable of identifying KSC and TA cells (and perhaps also cells that have been differentiated further) is used in place of xcex16xcex24 in the above two step process and that other marker might be another integrin such as xcex12xcex21 or xcex13xcex21 .
In a first aspect the invention could be said to reside in a method of enriching a viable population of KSCs from a population of epidermal cells comprising,
a) a first enriching step of enriching for cells carrying a high level of cell surface integrin from the population of epidermal cells to form a partially enriched pool, and
b) a second enriching step of removing cells that carry high level expression of a marker associated with proliferation from the partially enriched pool.
Conversely TA cells might be purified from KSCs whereby a proportion of cells with low expression of a marker associated with proliferation are removed from the partially enriched pool.
The epidermal cell population might be derived (torn a tissue sample of the skin. This method normally involves the separation of epidermis from the skin sample, before the enrichment. One particularly good source of KSC cells is from the basal layer of the epidermis. The proportion of these cells that are KSCs will depend upon the type of skin, and the age of the individual concerned. It is estimated for example that about 10% of neonatal foreskins are KSC cells but a lesser proportion will be present in the basal layer of the epithelium of adults. Hair follicles are also known to be a reservoir of stem cells and might be used as a source rich in KSCs.
It has been shown that significant enrichment can be achieved in the first step by the use of xcex21 in the first enrichment step, and it is thus thought that integrins xcex12xcex21 or xcex13xcex21 could be used in this enrichment. xcex21 is however less effective in the two step enrichment process than xcex16xcex24 integrin because it recognises epidermal cells that have developed past the TA stage and therefore the first enrichment step leads to a lesser enrichment than by use of xcex16xcex24 integrin which recognises only KSC cells and TA cells.
The marker associated with proliferation that has been used by the inventors is one that is recognised by monoclonal antibody 1007 and has now been identified as being the transferrin receptor. There are a number of commercially available monoclonal antibody preparations that also recognise transferrin receptor. Alternative markets that are associated with proliferation can also be used examples of these include but are not limited to the EGF (Epidermal Growth Factor) receptor, and perhaps also the IGF (Insulin Growth Factor) receptor and the KGF (Keratinocyte Growth Factor) receptor.
It will be understood recognition of cells carrying the cell surface markers that form the basis of the separation can be effected by a number of different methods, however, all of these methods rely upon binding of a binding agent to the integrin molecule, followed by a separation of chose that have high levels of binding from those that have low levels of binding. The most convenient binding agents are antibodies or antibody based molecules, preferably being monoclonal antibodies or based on monoclonal antibodies because of the specificity of these latter agents. Antibodies can be used for both steps. However other agents might also be used, thus ligands for these integrins such as extracellular matrix proteins including laminin-5 or collagen I or IV may also be employed to enrich for cells carrying xcex16xcex24. Likewise transferrin itself could be used as a means for detecting the levels of transferrin receptor, in place of 10G7 or other antibody directed against transferrin receptor.
The antibodies may be attached to a solid support to allow for crude separation. The separation techniques employed should maximise the retention of viability of the fraction to be collected. Various techniques of different efficacy may be employed to obtain relatively crude separations. The particular technique employed will depend upon efficiency of separation, associated cytoxicity, ease and speed of performance, and necessity for sophisticated equipment and/or technical skill. Procedures for separation may include, but are not limited to, magnetic separation, using antibody-coated magnetic beads, affinity chromatography and xe2x80x9cpanningxe2x80x9d with antibody attached to a solid matrix. Techniques providing accurate separation include but are not limited to, FACS.
In the experiments conducted thus far the xcex16xcex24 has been selected on the basis of the portion being identified, i.e. xcex16, however the xcex24 portion could equally well be used. Commercially available monoclonal antibody preparations that recognise xcex16 are available for these for example those known as GOH3 and 4F10.
It is found that by using an accurate separation method a purified KSC cell population can be achieved, which is believed to have less than 1% non KSC cells, however using cruder enrichment techniques a variety of levels of purified KSC cells can be produced, and may be useful at lower levels of purity. It is believed that purity levels of greater than 50% or at least greater than 70% may be useful for an improved formation of an epidermal layer which can be used as a graft for a skin lesion. A substantially purified KSC cell population of greater than about 90% is thought to be useful for not only the formation of an epidermal layer but also for use as a starting cell population for genetic modification whereby exogenous nucleic acid is introduced to express a desired product, which may be used in gene therapy.
In this regards it is considered that enriching for KSCs on the basis of the presence of xcex21 alone integrin will result in a population with only a limited capacity to provide a differentiated skin graft, it is suggested that selection of the xcex16xcex24 on its own may result in a sufficiently pure population of KSC cells to achieve an enhanced capacity to produce a differentiated epidermal layer. With a higher purity of KSCs it is expected that a graft should repopulate faster and should persist longer.
The capacity to isolate substantially purified KSCs opens up significant useful potential in some quite major areas.
Firstly autologous repair of skin lesions, by the formation of a layer of skin generated from cells isolated from the same individual. Methods of generating epidermis from epidermal cells are found for example in U.S. Pat. No. 5,712,163 by Parenteau et al which also refers to other references therein, particularly in column one which are incorporated herein by reference. It is suggested that these methods will be applicable where KSCs and TA cells of the present invention are used.
Secondly the accessibility of skin makes KSCs an ideal candidate for generic manipulation and gene therapy for the treatment of both skin disorders and systemic deficiencies. Thus exogenous nucleic acid would be introduced into autologous KSCs, to produce a therapeutically useful substance. The cells can be formed into an epidermal layer which could be grafted onto the skin of the individual concerned and act as a means for long term release of the therapeutic compound. The compound is thus introduced systemically. The compound might be one that is not produced by the individual as a result of a congenic defect, or a disorder that has developed, for example diabetes. Purification of KSCs according to the present invention are suggested to give rise to a skin graft of greater persistence and accordingly the gene therapy should last longer than would otherwise be possible. Suggestions for gene therapy using KSCs for gene therapy have been made with one of the acknowledged deficiencies being recognised as the lack of purified KSCs.
A third useful result of this invention relates to the enhanced capacity to find markers associated with proliferation of various cell sub populations of the skin involved in the early proliferative events and to enhance the understanding of these early proliferative events, with the greater potential to discover the reason for defects in the proliferative process which lead to cancers.
The invention could therefore also be said to reside in a composition including an enriched cell population of KSC cells capable of being enriched by firstly enriching a cell population for cells that carry an integrin marker and secondly reducing the number of cells that carry a marker associated with proliferation. Such compositions might include a tissue layer suitable for autologous skin graft application and a genetically modified population of KSC cells.
In a further form the invention could be said to reside in a composition formed from an enriched cell population of KSC of this invention. The composition could be the result of an epidermal layer that is used as a skin graft that has been derived from KSCs.